【作者】 殷翔；

【导师】 王爱民；

【作者基本信息】 第三军医大学 ， 外科学（专业学位）， 2013， 博士

【摘要】 研究背景：深静脉血栓形成是骨科临床常见并发症之一，好发于下肢骨折患者，如血栓脱落可引起致命性肺栓塞，晚期常遗留静脉炎综合征，严重影响患者的康复及生命安全。新近有研究表明富亮氨酸重复序列相互作用蛋白1（LRRFIP1）可能与血栓的形成有重要联系。LRRFIP1基因及其编码的蛋白质，通过与血小板细胞膜上表达的相关蛋白相互作用，血小板细胞骨架的结构进而发生改变，影响血小板的凝血功能并导致血栓的形成。因此我们拟通过RNA干扰技术靶向沉默LRRFIP1基因，并建立动物模型，观察LRRFIP1基因对深静脉血栓形成的影响，探讨LRRFIP1基因沉默对预防骨科深静脉血栓形成的效果，为最终应用于在体基因治疗深静脉血栓提供相关实验依据。研究方法：1、LRRFIP1基因RNAi慢病毒载体的构建设计并合成LRRFIP1基因靶向的双链DNA：根据GenBank报道的小鼠LRRFIP1基因序列，通过将Ambion公司的设计软件与RNA干扰序列的设计原则相结合，设计出3对针对LRRFIP1基因shRNA的寡核苷酸序列，分别构建质粒，转染293T细胞，RT-PCR和western blot测定沉默效率，使用沉默效率最高的质粒构建LRRFIP1基因RNAi的慢病毒载体。2、小鼠骨髓细胞原代分离与培养将1周龄的新生小鼠消毒后处死，分离骨髓细胞，接种于无菌培养瓶，置37℃、5%CO2恒温培养箱中培养。选取生长良好的第三代细胞进行后续实验。3、小鼠骨髓细胞慢病毒感染在24孔培养板接种若干孔，将3⁵X104个目的细胞接种至每个孔内，50%左右细胞的贴壁率应在铺板时达到，每孔培养基体积为100μl；70%左右的细胞贴壁率应在进行病毒感染时达到。感染96小时后，在荧光倒置显微镜下观察荧光，估计慢病毒感染目的细胞的效率。4、动物分组与给药将健康成年雄性ICR小鼠（20±2g）随机分为：1)正常对照组（control）；2)假手术组（sham）：不给药；3)低分子肝素（LMWH）治疗组（LMWH）：术前通过尾静脉注射LMWH一次(2000U·kg^-1)，术后每日注射一次LMWH (2000U·kg^-1·d^-1)；4)模型组（model）：术前、术后每日注射等体积生理盐水；5) LRRFIP1shRNA治疗组（shRNA）：术前注射LRRFIP1shRNA慢病毒感染的小鼠BMCs （1×10⁷cells/mouse）；6)阴性shRNA治疗组（Negative shRNA）：术前注射LRRFIP1阴性对照shRNA慢病毒感染的小鼠BMCs （1×10⁷cells/mouse）；采用下腔静脉结扎法制作小鼠深静脉模型。分别于术后第1、3、7天每组处死6只小鼠。称量血栓重量；酶联免疫吸附法（ELISA）检测小鼠血清p选择素及D二聚体水平，Western blot检测血栓中LRRFIP1蛋白的表达。5、统计分析计量资料表示为means±S.D。采用SPSS17.0for Windows进行统计分析，多组间比较采用单因素方差分析，多组间两两比较采用Fisher LSD分析。以p<0.05作为有统计学差异。结果：1.成功构建了携LRRFIP1shRNA的慢病毒载体，LRRFIP1shRNA转染后LRRFIP1基因及蛋白表达均显著降低；2. LRRFIP1shRNA显著抑制血栓中LRRFIP1蛋白表达。在模型组中，在术后第1、3、7天血栓中LRRFIP1蛋白的表达显著降低（P<0.01）。在所观察的3个时间点上，使用LRRFIP1shRNA处理可以进一步抑制LRRFIP1在血栓中的表达，提示LRRFIP1shRNA慢病毒在体内可以有效抑制LRRFIP1的表达。但低分子肝素和阴性shRNA对LRRFIP1表达无影响。3. LRRFIP1shRNA显著抑制小鼠下肢静脉血栓形成。LRRFIP1shRNA抑制体内血栓形成明显的。在术后第7天，模型组小鼠血栓重量为8.63±0.40mg，在LRRFIP1shRNA治疗组，血栓重量降低到2.15±0.57mg （P<0.01）。血栓形成抑制率为75.10%。而在低分子肝素治疗组和阴性shRNA治疗组，血栓重量分别为2.20±0.33mg和6.47±0.66mg，血栓形成抑制率分别为74.50%and25.02%。4. LRRFIP1shRNA能够降低小鼠血清p-选择素和d-二聚体水平。与正常对照组和假手术组相比，模型组血浆P-选择素水平明显增加（P<0.01）。LRRFIP1shRNA治疗显着降低血浆P-选择素水平（P<0.01）。血浆D-二聚体水平在模型组也明显升高（P<0.01），而LRRFIP1shRNA治疗可显著地抑制D-二聚体水平的上调（P<0.01）。然而，低分子肝素和阴性shRNA对血浆P-选择素、D-二聚体水平的影响不大。结论：1.成功构建了LRRFIP1shRNA表达质粒及慢病毒载体；2. LRRFIP1shRNA在体内、外均能够抑制LRRFIP1mRNA和蛋白表达；3. LRRFIP1基因干扰能抑制小鼠下肢深静脉血栓形成；4. LRRFIP1shRNA抑制深静脉血栓形成与其降低血浆p-选择素和d-二聚体水平有关。更多还原

【Abstract】 BackgroundDeep venous thrombosis （DVT） is one of the common complications in the departmentof orthopedics, occuring in the patients with fracture of lower limb. If the thrombi fall off, itcan cause fatal pulmonary embolism, and often left phlebitis syndrome, which seriouslyaffect the rehabilitation and life safety of patients. Recent studies have shown that leucinerich repeat interacting protein1（LRRFIP1） may have important links with thrombosis.LRRFIP1gene and its encoded protein, may interact with the protein on platelet membrane,then change the platelet cytoskeleton structure, and affect the coagulation function inplatelet and thrombus formation. So we proposed to observe the effect of LRRFIP1gene onthe formation of DVT through silencing LRRFIP1by RNA interference, investigate theeffect of LRRFIP1gene silencing on the prevention of DVT and provide the experimentalbasis for the final application of gene therapy in the treatment of DVT in vivo.Methods1. Construction of lentiviral vector carrying LRRFIP1shRNADesign and synthesize LRRFIP1gene targeted double-strand DNA: according to therat LRRFIP1gene sequence reported by GenBank, using the design software of Ambioncompany, according to RNA interference sequence design principles, design3againstLRRFIP1gene shRNA oligonucleotide sequence. Construct and transfect the plasmids into293T cells, RT-PCR and Western blot methods determine the silencing efficiency. Themost efficient shRNA plasmid was used to package the lentiviral vectors.2. Isolation and culture of the primary mouse bone marrow cells （BMCs）One-week old rats were sacrificed after disinfection. BMCs were isolated andincubated in37C,5%CO2incubator. P3cells were selected for the followingexperiments. 3. Infection of BMCs with lentivirusBMCs were seeded in24-well plates at a density of3⁵X104/well. When theconfluence reached70%, the BMCs were infected with the lentivirus. After96h, thefluorescence was observed under the fluorescent microscope, and the infection efficiency ofthe lentivirus was estimated.4. Animal grouping and treatmentHealthy adult male ICR mice （20±2g） were randomly divided into the followinggroups:1) normal control group （control）;2) the sham operation group （sham）;3) lowmolecular weight heparin （LMWH） treatment group （LMWH）: mice were injected withLMWH (2000U kg^-1) once by tail vein preoperatively, and postoperatively injected withLMWH (2000U kg^-1 d^-1) daily;4) model group （model）: mice were injected with normalsaline daily;5) LRRFIP1shRNA treatment group （shRNA）: mice were injected with BMCsinfected with LRRFIP1shRNA lentivirus before operation （1×10⁷cells/mouse）;6)negative shRNA treatment group （Negative shRNA）: mice were injected with BMCsinfected with negative LRRFIP1shRNA lentivirus before operation （1×10⁷cells/mouse）.DVT mouse model was produced by ligation of inferior vena cava. Six mice in eachgroup were sacrificed on the1st, the3rdand the7thday post operation. The weight ofthrombus was determined; enzyme linked immunosorbent assay （ELISA） detection wasused to examine serum P-selectin and d-dimer levels; expression of LRRFIP1protein inthrombosis was determined by Western blot.5. Statistical analysisMeasurement data were expressed as means±S.D. SPSS17for Windows was usedfor statistical analysis. Statistical analysis was programmed by one-way analysis ofvariance （ANOVA）. Difference was regarded as significant when P <0.05.Results1. The lentiviral vector carrying LRRFIP1shRNA was successfully constructed.transfection of LRRFIP1shRNA significantly decreased LRRFIP1mRNA and proteinexpression;2. LRRFIP1shRNA significantly inhibited the expression of LRRFIP1protein inthrombus. In the model group, the expression of LRRFIP1in thrombus decreasedsignificantly （P<0.01） on the1st,3rdand7th days after operation. At the3time points observed, LRRFIP1shRNA treatment further suppressed LRRFIP1expression in thrombus,indicating LRRFIP1shRNA lentivirus can effectively inhibit the LRRFIP1expression. Butthe LMWH and negative shRNA had no effect on the expression of LRRFIP1.3. LRRFIP1shRNA significantly inhibited mice DVT formations. LRRFIP1shRNAinhibited thrombus formation in vivo apparently. On the7th day post operation, the weightof thrombus in model group was8.63±0.40mg, whereas the thrombus weight reduced to2.15±0.57mg （P<0.01） in the LRRFIP1shRNA treatment group, with a thrombosisinhibition rate of75.10%. In the LMWH treatment group and shRNA negative group, thethrombus weight were2.20±0.33mg and6.47±0.66mg, with thrombosis inhibition ratesof74.50%and25.02%respectively.4. LRRFIP1shRNA can reduce the serum levels of p-selectin and d-dimer. Comparedwith the normal control group and sham operation group, the plasma level of P-selectinsignificantly increased （P<0.01） in model group,. LRRFIP1shRNA treatment significantlydecreased the plasma level of P-selectin （P<0.01）. The plasma level of d-dimer in modelgroup was also significantly increased （P<0.01）, and LRRFIP1shRNA treatmentsignificantly inhibited the up-regulation of d-dimer levels （P<0.01）. However, LMWH andnegative shRNA showed little effect on plasma P-selectin and d-dimer levels.Conclusion1. The LRRFIP1shRNA expression plasmid and lentiviral vector were successfullyconstructed;2. LRRFIP1shRNA is able to inhibit LRRFIP1mRNA and protein expression in vitroand in vivo;3. LRRFIP1gene knockout inhibits deep venous thrombosis formation in mice;4. Inhibition of deep vein thrombosis with LRRFIP1shRNA is associated with thedecrease of plasma p-selectin and d-dimer levels.更多还原